Peptidic Bioactivable Au Nanoclusters for Extracellular Vesicles and Cellular Labelling and Biosensing

We present an overview of the interaction between ultrasmall luminescent gold nanoclusters and lipidic or biological membranes, showing that it is possible to induce a large change in membrane structures or not. The synthesis of original aqueous bioactivatable and PEGylated AuNC suspensions of various charges were performed by using synthetic short peptides. This chemical plateform provides various charged AuNC with potential sensitivity to the environment for biosensing applications.<br/>Extracellular vesicles (EVs) are well-known membrane-limited particles that are secreted by healthy and cancerous cells. EVs are heterogeneous in size and three subtypes are described depending on the location of secretion: microvesicles, myelinosomes and exosomes. EVs are identified in human follicular fluid as a mode of communication in the ovarian follicle (Neyroud A. S. et al. Int. J. Molecular Sci. <b>2022</b>). In addition EVs involved in cell-cell communication are considered as biomarkers for early cancer diagnosis. The analysis of their content and their labeling with easily detectable nanoparticles could enable the development of a powerful tool for the early diagnosis of specific diseases.<br/>In this view, Gold nanoclusters (AuNCs) appear as a recent class of non-toxic fluorophores. Their brightness, their ultrasmall size (&lt; 2 nm) and large window of fluorescence lifetime (1ns – 1ms) and their good biocompatibility make them an attractive alternative as fluorescent probes for biological labeling and bioimaging.<br/>Following incubation of the AuNCs with oppositely charged vesicles, either liposomes or EVs, the strong electrostatic attraction resulting in the adsorption of AuNCs to the membranes was evidenced by complementary techniques such as zetametry, fluorescence optical microscopy, SAXS, and cryo-TEM. In the presence of an excess of oppositely charged AuNCs, the liposomes strongly adhere to each other without disrupting their membrane whereas the EVs extracted from human follicular fluid rearrange into a hybrid lamellar phase. Instead, in the presence of a membrane surface excess, AuNCs do not change the size of the membrane thickness, so they are positioned between the polar headgroups of the membrane phospholipids. As opposed to larger gold nanoparticles, the smaller size of AuNCs not only prevents the deformation of biological membranes but also allows labeling with higher spatial resolution (R. Cheichio et al. J. Phys. Chem. Lett <b>2022</b>).<br/>By adjusting the surface ligands, these probes are easily internalized into cells and in vivo organisms such as Arabidopsis plant or cancerous cell lines. We demonstrated their in vivo targeting ability because of specific recognition groups (R. Cheichio et al. ACS Nano materials <b>2023</b>) and biosensing by fluorescence because of their sensitivity to microenvironment.<br/>In addition their small size make them attractive to encapsulate them into liposomes without damaging the compartment integrity and then to be delivered into the extracellular vesicles. We demonstrated first the possible encapsulation of the AuNC into liposomes for drug delivery. Cell-like-sized vesicles (GUVs) encapsulating red or blue Au NCs were successfully obtained by an innovative method using emulsion phase transfer. Finally, exosome-like-sized vesicles (LUVs) containing Au NCs were obtained with an encapsulation yield of 40%, as estimated from ICP-MS (R. Chiechio et al. Nanomaterials <b>2022</b>). Finally the lipidic membranes composition was optiized with ionizable lipids so that the mixture of the liposomes and EVs results in efficient fusion and delivery of AuNC into the lumen of EVs. The lipid exchange was demonstrated by FRET experiments and fusion was revealed by the increased size and the fluorescent labelling of the fused EVs. The fused EVs encapsulating the AuNC were successfully separated by size exclusion chromatography. Such nanostructures offer promising candidates for fluorescent in vivo biosensing and biolabeling.